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Final Report Summary - TOPBIO (Two Photon Absorbers for Biomedical Applications)

TOPBIO overview and objectives
The TOPBIO project aims at providing an integrated training from both academic (8 partners) and industrial groups (3 partners) in the emerging field of two-photon absorption (2PA), focusing on the applications offered by the development of two-photon excitation technologies in the biological and biomedical arena. These include cell biology (microscopic bioimaging, photo-activated drugs), photodynamic therapy and biomedical engineering (3D microfabrication of biomedical devices). This implies the design, preparation and application of specifically tailored new dyes that are highly responsive to two-photon excitation, as well as fine understanding of biological constraints and the use of optimized optical sources and instrumentation. The TOPBIO project thus relies on interdisciplinary training ranging from synthetic chemistry to physical and/or polymer chemistry, and from cellular biology to lasers and nanotechnology. This involves:
• Theoretical approaches for fundamental understanding of the molecular design criteria for optimizing TPA in order to design better two-photon dyes allowing faster operations or the use of lower excitation intensity (theoretical chemistry);
• Implementation of unified and reliable experimental protocols for the accurate determination of the two-photon absorption responses of two-photon dyes, allowing for the evaluation of the new dyes and consistent comparison with already available standard dyes (optics and lasers);
• Design, synthesis and photophysical characterization of advanced two-photon dyes for either two-photon imaging for cell biology, two-photon uncaging of drugs or bioactive compounds, two-photon photodynamic therapy, or two-photon microfabrication of medical implants or scaffolds for tissue engineering (molecular engineering, organic synthesis, photophysics);
• Application in bioimaging, photodynamic therapy (investigation of photo-induced cell death mechanisms), photorelease of bioactive drugs (cellular biology, neurophysiology);
• Microfabrication for biomedical applications (polymer chemistry, engineering, nanotechnology).

Main results achieved so far
The TOPBIO project has already advanced the state-of-the-art in several of these directions as a result of a unified theoretical and experimental approach and favorable synergic interactions between partners favored by Early Young Researchers (ESRs) efforts and exchanges:
• Fundamental understanding of the molecular design criteria for optimizing 2PA: Challenging computational methods (high level ab initio) are used to examine and model the 2PA response of complicated molecules and taking into account surrounding environment (including protein encased-chromophores). This is a key issue as long as bio-applications are concerned (two-photon imaging and photodynamic therapy) due to the high sensitivity of TPA to environmental effects.
• Development of practical techniques for accurately characterizing TPA spectra: These include the development of an experimental set-up for measuring absolute 2PA cross- section values for a selection of 2PA standards using the two-photon induced fluorescence technique (2PEF). For non-fluorescent two-photon novel dyes (such as those specifically engineered for two-photon microfabrication by 2P-induced polymerization or photodynamic therapy), an experimental set-up has been developed to allow measurements of 2PA coefficients, using the Z-scan technique. The combination of the two techniques has allowed accurate measurements and comparison of the novel 2P dyes produced within the TOPBIO consortium since the beginning of the project, allowing fast feedback loops and providing a major support for the design of “second generation” optimized 2P dyes for each application.
• New 2P dyes for two-photon excited fluorescence microscopy: Several innovative approaches have been instigated in order to obtain fluorescent dyes showing large 2PA in the biological spectra window (700-1000 nm). In particular successful routes have been implemented to enhance 2PA in porphyrin derivatives (either by introducing strong electron-withdrawing peripheral substituents or by engineering the conjugation in “tape” dimers). This lead to porphyrin dimers combining giant 2PA response (larger than 7000 GM), NIR emission and reasonable fluorescence quantum yield (10%) in organic environment. In addition, libraries of novel nonporphyrinoid derivatives possessing intrinsically polar building blocks (expended imidazoles, imidazopyridines) have been synthesized and investigated, yielding new routes for 2PA optimization.
• Two-photon induced cell death: An extracellular sensitizer has been successfully used to induce cell death by two-photon excitation, allowing quantification of the light dose required for initiating cell death and opening the way to the elucidation of photo-induced cell death mechanisms.
• Two-photon polymerization: Novel biodegradable conducting and hybrid photostructurable materials have been investigated for their suitability as scaffolds for tissue engineering and polymer-based nanogrids for use as a substrate for cells have been developed.

Outlooks: The on-going progress of the TOPBIO project hold many promises in the field of biomedical applications, of potential large benefic for the community. For example, elucidating spatially-dependent mechanisms of photosensitized cell death could lead to the development of more efficient drugs for use in cancer photodynamic therapy. Novel fluorescent dyes combining giant two-photon absorption and retaining fluorescence in bioenvironment will be of high interest for high resolution in vitro to in vivo imaging. In case of success, this would pave the way to easier and more sensitive diagnosis of various diseases and whole societies will benefit from this fact. The same holds for the production of biomedical microdevices (implants, delivery systems, tissue scaffolds) thanks to new 2PA photoinitiators of polymerization.
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